EXPERIMENTAL AND MOLECULAR PATHOLOGY 52,25-36 (I!?.@) Changes in Myofibrillar Components after Skeletal Muscle Necrosis Induced by a Myotoxin Isolated from the Venom of the Snake Bothrops asper Jo& MARIA GUTIBRREZ,’ VILMA ARCE,* FERNANDO BFENES,~ AND FERNANDO CHAVES’ ‘Institute Clodomiro Picado, Facultad de Microbiologia, and ‘Centro de Znvestigacibn en Biologia Celular y Molecular, Universidad de Costa Rica, San Jose, Costa Rica; and ‘Laboratorio de Patologia Experimental, Hospital Mkico, Caja Costarricense de1 Seguro Social, Costa Rica Received May 12, 1989, and in revised form August 28, 1989 The effects of a myotoxic phospholipase A, isolated from the venom of the crotaline snake Bothrops asper on skeletal muscle myotibrils were studied by histological, ultrastructural, immunohistochemical, and biochemical parameters. Myotoxin induced a rapid and promi- nent muscle necrosis after intramuscular injection in mice. In this process, myofibrils were affected and three main changes were observed: (A) Initially, they were hypercontracted, eventually forming “clumped,” dense masses which alternated with spaces devoid of my- ofilaments in the cytoplasm. This initial stage is probably due to hypercontraction resulting from a calcium influx after toxin-induced sarcolemmal damage. (B) A second change oc- curred between 3 and 6 hr, when the clumped or hypercontracted pattern changed to a “hyaline” pattern in which myotilaments were relaxed and had a more uniform distribution in the cellular space. Although there was not a widespread degradation of myofibrillar components at this stage, desmin started to be lost in samples obtained as early as 15 min after toxin injection, and a-actinin was almost absent by 7 hr. Thus, it is proposed that this shift may be due to a selective proteolytic degradation of structurally relevant components, particularly a-a&tin. As a consequence, the mechanical integration of myofdaments is impaired, precluding hypercontraction. (C) Finally, at later time periods (24,48, and 72 hr), there was widespread degradation of myotibrillar proteins, probably caused by proteases derived from intlanunatory cells such as neutrophils and macrophages, whose numbers in necrotic muscle increased markedly at these time periods. Q EJW Academic has, hc. INTRODUCTION Many snake venoms induce skeletal muscle necrosis clinically as well as in experimental animals (Tu, 1977). Some of them contain myotoxins, i.e., proteins that directly damage muscle cells (Harris et al., 1980; Gutierrez et al., 1984a), whereas in other cases muscle damage is indirect, being due to the ischemia which develops as a consequence of microvessel damage (Gleason et al., 1983; Gutierrez et al., 1984b). In both cases myolibrils are drastically affected as a consequence of venom action. However, little is known about the detailed morphological and biochemical changes occurring in myolibrils after injection of snake venoms and toxins. A basic myotoxic phospholipase A2 was isolated from the venom of the Central American pit viper Bothrops usper (Gutierrez ef al., 1984a), and has recently been named Bothrops usper myotoxin I (Lomonte and Gutierrez, 1989). This toxin induces muscle damage by affecting initially the plasma membrane (Gutierrez et al., 1984b; Brenes et al., 1987). After this initial alteration, there is a rapid se- quence of degenerative events in myotibrils, mitochondria, sarcoplasmic reticu- lum, and nuclei, a process that rapidly ends in irreversible cell damage. Other myotoxic agents such as tarantula venom (Ownby and Odell, 1983), bupivacaine 0014-4800190 $3.00 Copyright 0 1990 by Academic Press, Inc. AU rights of reproduction in any form reserved.